Moderate Caloric Restriction Suppresses microRNAs miR-696 and miR-199a in a Voluntary Running Wheel Model

Abstract

The transcriptional coactivator PGC-1α is central to skeletal muscle adaptation to increased physical activity. SIRT1, an energy sensing deacetylase, and important regulator of transcription, plays a role in the benefits of caloric restriction. SIRT1 is also a regulator of PGC-1αin skeletal muscle. MicroRNAs (miRNAs) are short noncoding RNA molecules involved in post-transcriptional regulation of gene expression by binding to target mRNA. Expression levels of miRNAs can be altered by physiological stimuli. miR-696 and miR-199a are reported to target PGC-1αand SIRT1, respectively. Neither expression of miR-696 nor miR-199a has been studied in the adaptation of skeletal muscle to physical activity and moderate caloric restriction (CR). PURPOSE: We tested the hypothesis that moderate CR in physically active mice would suppress expression of miR-696 and miR-199a. METHODS: Male C57BL/6J mice (9 wks old) were individually housed and assigned to one of two groups: ad libitum feeding (AL, n=7) or moderate CR (n=6). The AL mice had free access to food and a running wheel for 23 days. The moderate CR mice had free access to a running wheel for 23 days and free access to food for the initial 8 days, and then moderate CR for 15 days. The moderate CR protocol was implemented to achieve a 20% reduction in body mass within 3 days. Body mass reduction was then maintained for the remainder of the experiment. Running distance was measured daily. Total RNA was isolated from the plantaris (PLA). Total RNA and microRNA were reverse transcribed in separate reactions to measure expression levels of COXI, PGC-1α, and SIRT1, or miR-696, miR-199a. The relative copy number of mitochondrial (mt) DNA was measured in total DNA (nuclear and mitochondrial) extracted from PLA. RESULTS: The moderate CR group required a 26% decrease in food intake to achieve the 20% decrease in body mass. During the 15 days of moderate CR mice ran 10.5 km/day while the AL mice ran 6.9 km/day (p<0.05). mtDNA copy number and COXI expression were significantly (p<0.05) greater, 27% and 64% respectively, in moderate CR mice, while PGC-1αexpression was not affected (p>0.05). However, miR-696 expression was suppressed (70%, p<0.05) in moderate CR mice. Moderate CR resulted in increased SIRT1 expression (66%, p<0.05) and conversely, decreased miR-199a expression (58%, p<0.05). CONCLUSION: Compared to wheel running alone, moderate CR significantly increased running wheel distance, COX1 and SIRT1 mRNA expression, mtDNA copy number, and suppresses microRNAs miR-696 and miR-199a in physically active mice. These data suggest that moderate CR may regulate mitochondrial biogenesis through post-transcriptional mechanisms.